The Epidermal Growth Factor Receptor (EGFR, also called ErbB1 or HER1) belongs to the family of single transmembrane domain receptors with tyrosine kinase activity driven by their intracellular domain. The EGFR family also includes ErbB2, ErbB3 and ErbB4 receptors. The extracellular domain of these receptors presents a ligand-binding site. The binding of a specific ligands induce the receptor activation, ultimately triggering signal transduction pathways involved in cell proliferation, migration, differentiation and survival (1). In addition to EGF itself, 11 different ligands have been shown to activate those receptors (including TGF-α, β-Cellulin and Neuregulins) (2). ErbB1 is activated by all ligands of this family except Neuregulins, specific for ErbB3 and ErbB4. These ligands bind to the extracellular domain of the receptor, inducing a major conformation change. This new conformation allows the formation of homodimers, or induce the formation of heterodimers with other members of the family (3). The structural basis for ligand-induced dimerization of ErbB extracellular regions is now well understood (4-9), and leads to an allosteric activation of the intracellular tyrosine kinase domain. However, although ligand binding and dimerization events seem connected, several studies have demonstrated that EGFR can also be found in the cell surface as non-activated dimers, also called predimers (10-13). The ligand-independent EGFR/ErbB2 dimer formation was shown to require the cytoplasmic domain of EGFR to be present on resting cells (14, 15). The relationship between extracellular region, transmembrane domains, intracellular juxtamemembrane domains and cytoplasmic tyrosine kinase of ErbB family receptors, plays a major role in the dimerization and activation events (16). Moreover, conformational changes during these activations appear to be key for the signal transduction. Because these inactive predimers are proposed to be primed for ligand binding and allow a fast and efficient signaling, they might represent a very relevant therapeutic target for small inhibitors. Unfortunately, despite the large amount of structural and functional data concerning the various states of EGFR, the conformation of its extracellular domain within predimers has never been captured by crystallography studies and remains largely unknown. Therefore, a precise monitoring of EGFR conformational changes appear crucial to thoroughly understand EGFR family signaling and help in the design of small-molecule drugs. Beside crystallography, other powerful methods such as nuclear magnetic resonance (17) or electron microscopy are increasingly used to solve high quality structures (18) but remain cumbersome and cannot be used on intact cells. Other methods are based on the use of mutant or fusion proteins (19, 20) but they also cannot be used on cells naturally expressing the wild type receptor. Molecular dynamics simulations (21) can provide essential insights but as any in silico prediction tools, it requires an experimental validation of the findings.
Recently, the use of non-conventional antibodies has emerged as a simple, new and sensitive approach to study protein conformation on living cells. Single domain antibodies (sdAbs, also called nanobodies) (22), correspond to the variable domains of a special class of antibodies naturally devoid of light chains found in Camelids. These small proteins (15 kDa) present several advantages (23) including a good thermal stability even without disulfide bond formation (24), a good solubility and high expression yield (25). Most importantly, sdAbs have a natural tendency to bind epitopes that are inaccessible to conventional antibodies (26), such as cleft and cavities. Consequently, they are often very sensitive to conformational changes of their target (27, 28). However, anti-Epidermal Growth Factor Receptor (EGFR) conformational single domain antibodies are still needed.